CCNU given i.v. to male Fischer rats is rapidly hydroxylated on the cyclohexyl ring to give at least five metabolites. When exposed to alkaline pH at 100 degrees CCNU and its metabolites quantitatively release their cyclohexyl moiety as cyclohexylamine and aminocyclohexanol, respectively. The N-(2,4-dinitrophenyl) derivatives of cyclohexylamine and aminocyclohexanols were separated by high pressure liquid chromatography. The in vivo metabolites have been identified as trans-2-hydroxy CCNU, cis-3-hydroxy CCNU, trans-3-hydroxy CCNU, cis-4-hydroxy CCNU and trans-4-hydroxy CCNU. Ring hydroxylation axial to the 1-(2-chloroethyl)-1-nitrosourea group (cis-2-, trans-3-, cis-4-) is favored over equatorial attack (trans-2-, cis-3-, trans-4-). Pretreatment of rats with phenobarbital leads to an increased rate of hydroxylation and a change in the relative amounts of the hydroxylated products. Plasma samples from several patients receiving CCNU chemotherapy have been analyzed for parent drug and hydroxylated metabolites up to 8 hours following therapy. The major portion of the plasma nitrosourea is always present as hydroxylated metabolites which have been identified as cis-4-hydroxy CCNU and trans-4-hydroxy CCNU. A study of tissue distribution of CCNU and its metabolites is planned with a view to determining the pharmacologic and therapeutic consequence of CCNU metabolism.